Field of the Invention
The present invention relates to a method and apparatus for improving the expansion of stem cell populations. More particularly, the present invention relates to a new bioreactor and method for expanding mesenchymal stem cells in culture.
Description of the Related Art
Mesenchymal stem cells (MSCs) are adult stem cells traditionally found in the bone marrow. However, MSCs can also be isolated from other tissues including adipose tissue, umbilical cords, placenta, and dental pulp. Preliminary experimental results indicate that adult mesenchymal stem cells (MSCs) may provide therapeutic and/or regenerative benefits in the treatment of certain diseases. Such results have fueled a considerable excitement in the scientific and health communities regarding the potential of stem cells to treat a number of disease states. In fact, there are a number of clinical trials currently in progress in an effort to ascertain the true therapeutic potential of MSCs.
Originally the scientific community thought that the therapeutic benefits of MSCs resulted from the engraftment and differentiation of MSCs to replace diseased cells. However, subsequent data indicates that the beneficial effects of MSCs occur without evidence of engraftment. Rather, it has been shown that MSCs enhance tissue repair through secretions and cell-to-cell contacts (Prockop D. J. “Repair of tissues by adult stem/progentor cells (MSCs): Controversies, myths and changing paradigms”, Mol. Ther. 17: 939-946, 2009; Sotiropoulou P. A., et al. “Interactions between human mesenchymal stem cells and natural killer cells”, Stem Cells 24: 74-85, 2006).
A major problem in the successful clinical use of MSCs, as compared to pluripotent stem cells, is that MSCs have a low capacity to replicate ex vivo and must be continuously harvested from donors and expanded in culture to produce enough cells for therapeutic applications. In addition, as MSCs are passaged, they undergo a change in phenotype and diminished therapeutic efficacy (Izadpanah R., et al. “Long-term in vitro expansion alters the biology of adult mesenchymal stem cells”, Cancer Res. 68: 4229-4238, 2008). The differentiation and diminished efficacy of passaged MSCs continues to impose severe limits on the ex vivo expansion of therapeutically potent MSCs.
Problems with the self renewal of stem cells in culture are apparently related to the fact that the conventional cell culture environment does not resemble the in vivo environment in which the stem cells normally exist. In vivo, stem cells are maintained in a largely quiescent state. Most cell divisions lead to the production of a stem cell and a daughter cell which will continue to divide to eventually produce a tissue. The numbers of stem cells actually change very little. This situation is thought to have evolved to minimize the possibility of mutations which could lead to uncontrolled growth or cancer.
Currently, the commercial expansion of MSCs is done in multilevel modules such as the Nunc® Cell Factory™ or the Corning HYPERStack®. These devices are merely high surface area analogs of standard tissue culture flasks. While these multilevel modular devices are capable of growing a large numbers of MSCs, they do not prevent the differentiation and diminished efficacy normally seen in culturally expanded MSCs. However, recent studies have shown an enhanced therapeutic potency for MSCs cultured in three dimensions as compared to MSCs expanded using conventional two dimensional culture systems (Frith, J. E., et al. “Dynamic three-dimensional culture methods enhance mesenchymal stem cell properties and increase therapeutic potential”, Tissue Eng. Part C Methods 16: 735-749, 2010). This reported improvement in therapeutic potential is likely due to the increased secretion of paracrine factors and the reduced cell size which permits infused MSCs to migrate to an injury site through the smallest arterioles (Bartosh, T. J., et al. “Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties”, Proc. Natl. Acad. Sci. (USA) 107: 13724-13729, 2010).
It has been proposed that culturing MSCs in three dimensional culture systems more closely resembles the in vivo microenvironment, or niche, of these cells than the two dimensional systems. Simulating the normal microenvironment of the cells in vivo is thought to be a critical factor in maintaining the MSC phenotype and therapeutic potential during cell expansion (Cheng, N-C, et al. “Short-Term Spheroid Formation Enhances the Regenerative Capacity of Adipose-Derived Stem Cells by Promoting Stemness, Angiogenesis and Chemotaxis”, Stem Cells Translational Med. 2: 584-594, 2013).
An ongoing problem in the three dimensional culturing of MSCs is that there are no successful large-scale commercial MSC culturing systems available. All of the initial work done on three dimensionally cultured MSCs has been done using spheroids produced by hanging drop culture or hydrogel encapsulated cells, techniques which are inherently limited in the scale of MSC production. Thus, a continuing need exists for improving three dimensional culture systems for the large scale expansion of stem cells.